Embodiments of the present invention relate to the field of nucleic acid sequencing, and more specifically, embodiments of the present invention provide methods, systems and devices that utilize a plurality of excitation wavelengths to image multiple emission patterns through an optical detection path comprised of stationary components.
Numerous recent advances in the study of biology have benefited from improved methods for analyzing and sequencing of nucleic acids. For example, the Human Genome Project has determined the entire sequence of the human genome which is hoped to lead to further discoveries in fields ranging from treatment of disease to advances in basic science. Devices for DNA sequencing based on separation of fragments of differing length were first developed in the 1980s, and have been commercially available for a number of years. However, such technology involves running individual samples through capillary columns filled with polyacrylamide gels and is thus limited in throughput due to the time taken to run each sample. A number of new DNA sequencing technologies have recently been reported that are based on the massively parallel analysis of unamplified, or amplified single molecules, either in the form of planar arrays or on beads.
The methodology used to analyze the sequence of the nucleic acids in such new sequencing techniques is often based on the detection of fluorescent nucleotides or oligonucleotides. The detection instrumentation used to read the fluorescence signals on such arrays is usually based on either epifluorescence or total internal reflection microscopy. One detection instrument has been proposed that use an optical sequencing-by-synthesis (SBS) reader. The SBS reader includes a laser that induces fluorescence from a sample within water channels of a flowcell. The fluorescence is emitted and collected by imaging optics which comprise one or more objective lens and tube lens. As the fluorescence travels along an optics path within the imaging optics, but prior to reaching a detection camera, the fluorescence propagates through an interference emission filter. The emission filter has the ability to select wavelength bands of interest from the fluorescence and block other wavelength bands that are associated with noise, such as laser scatter or the emission from orthogonal fluorophores that emit at different wavelengths.
One conventional approach to performing spectral splitting of fluorescence is to use bandpass filters in conjunction with an emission filter wheel, where an emission filter wheel is located along the optical path before each detection camera. The emission filter wheel is a mechanical device that is rotated, under control of a servo motor, until an appropriate filter is placed in the optical path of the fluorescence. However, the use of filter wheels and servo motors in the detection path is not always desirable. As an example of the use of a mechanical filter wheel in operation, a high throughput sequencing instrument that is capable of capturing an image every few hundred milliseconds, means that in the course of a single days use, the filter wheel is used hundreds of thousands of times. During operation, the filter wheel is mechanically rotated between imaging cycles which introduces complexity and a filter switching time that reduces the overall operation rate of the detection system. Also, because the filter wheel is a mechanically moving element, it and other moving elements will have a limited life span and may introduce error over time as the elements wear. Finally, care is needed to properly align and calibrate the filter wheel.
There is a continuing need for better, more robust, and more economical devices and systems for fast reliable sequencing of nucleic acids. Embodiments of the present invention seek to address these needs and offer other benefits which will be apparent upon examination of the current specification, claims, and figures.